Nona-peptide and deca-peptide analogs of LHRH, useful as LHRH antagonists

ABSTRACT

Synthetic nona-peptide and deca-peptide LHRH antagonist analogs have a novel guanido-substituted, amidine, tertiary or quaternary aminoacyl residue at position 6.

This application is a continuation in part of pending U.S. patentapplication Ser. No. 451,671, filed Dec. 21, 1982, U.S. Pat. No.4,481,190.

BACKGROUND OF THE INVENTION

Luteinizing hormone (LH) and follicular stimulating hormone (FSH) arereleased from the anterior pituitary gland under the control of thereleasing hormone LHRH produced in the hypothalamic region. LH and FSHact on the gonads to stimulate the synthesis of steroid hormones and tostimulate gamete maturation. The pulsatile release of LHRH, and therebythe release of LH and FSH, controls the reproductive cycle in domesticanimals and humans.

LHRH also affects the placenta, and the gonads indirectly, in causingthe release of chorionic gonadotropin (hCG).

Antagonists of LHRH are useful for the control of fertility. Suchantagonists block ovulation in the female and suppress spermatogenesisin the male. Related to these effects is a suppression of normalcirculating levels of sexual steroids of gonadal origin, includingreduction in accessory organ weight in the male and the female. Indomestic animals this effect promotes weight gain in a feed-lotsituation, stimulates abortion in pregnant animals and in general, actsas a chemical sterilant.

The natural hormone releasing hormone LHRH is a decapeptide comprised ofnaturally occuring amino acids (which have the L-configuration exceptfor the achiral amino acid glycine). Its sequence is as follows:##STR1## Many analogs of this natural material have been studied and thevery large majority of them have proven to be of insufficient biologicalactivity to be clinically useful. Certain select modifications haveproven to have an agonist effect on biological activity. By far the mostsignificant enhancement is obtained by changing the 6-position residuefrom Gly to a D-amino acid.

In addition to agonists, analogs have been prepared which arecompetitive antagonists to LHRH; all of which require deletion orreplacement of the histidine residue at position 2; Vale, W., et al,Science, 176: 933 (1972). In general, it appears that a D-amino acidplaced in the sequence at that position gives the best activity; Rees,R. W. A., et al, J. Med. Chem. 17: 1016 (1974).

It has also been shown that adding a modification at the 6 position,which, without the modification at position 2, results in the agonistactivity cited above, enhances the antagonist activity of the 2-modifiedanalogs; Beattie, C. W., et al, J. Med. Chem., 18: 1247 (1975); River,J., et al, Peptides 1976 p. 427, Editions de l'Universite de Bruxelles,Belgium (1976).

Against the background of these two major alterations, which result in apotent series of LHRH antagonists; additional increments in antagonistactivity may be had by modifying positions 1, 3 and/or 10 in the already2, 6 modified peptide. Coy, D. H., et al Peptides 1976, p. 462, Editionsde l'Universite de Bruxelles, Belgium (1976); Rivier, J. E., et al, LifeSci. 23: 869 (1978); Dutta, A. S., et al, Biochem Biophys. Res. Commun.81: 382 (1978), Humphries, J., et al, Biochem. Biophys. Res. Commun.,85: 709 (1978). It has also been shown that N-acylation of the aminoacid at position 1 is helpful; Channabasavaia, K., et al, Biochem.Biophys. Res. Commun. 81: 382 (1978); Coy, D. H., et al, Peptides. -Structure and Biological Function p. 775, Pierce Chemical Co. (1979).Additionally, (N-Ac-D-p-Cl-Phe¹, D-p-Cl-Phe², D-Trp³, D-Arg⁶,D-Ala¹⁰)LHRH has been published by D. H. Coy, Endocrinology, 110, 1445(1982). In another instance D-Ala⁴ modification to LHR has been reportedto retain antagonist activity. See E. Pedroza, J. A. Martinez, D. H.Coy, A. Arimura and A. V. Schally; Int. J. Fert.; 23, 294 (1978).

Since antagonists function by competing with LHRH for the appropriatereceptors, high dosages of these compounds are required in order toblock out the natural peptide. It is especially desirable, in view ofthis, to obtain antagonists with a very high degree of potency andprolonged activity. The ability to be slowly released from depotformulations will also be important. The presently known set of analogsrequires comparatively high levels of compound, with the attendantproblems of increased possibility for toxicity and other side effects.

SUMMARY OF THE INVENTION

The present invention refers to novel, highly potent nonapeptide anddecapeptide analogs of LHRH in which a replacement at position 2, (thusconverting the peptide to the antagonist series) is made more effectiveby replacement of the glycine residue at position 6 by a novelguanido-substituted, amidine, or tertiary or quaternary amine watersoluble amino acid residue which does not occur in nature. Furtherenhancements by substitutions at 1, 2, 3, 4, 7 and/or 10 are alsodisclosed. The invention is also directed to various methods of use ofthese compounds and to pharmaceutical compositions therefor. A furtheraspect of the invention involves processes for the preparation of thenovel compounds described above.

DETAILED DESCRIPTION OF THE INVENTION Description of the Analogs

The present invention relates to novel nonapeptide and decapeptideanalogs of LHRH which have the formula ##STR2## and the pharmaceuticallyacceptable salts thereof, wherein: A is an amino acyl residue selectedfrom the group consisting of N-Ac-D,L-Δ³,4 -prolyl, N-Ac-D,L-prolyl,N-Ac-L-alkylprolyl, N-Ac-D,L-phenylalanyl,N-Ac-D,L-p-chlorophenylalanyl, N-Ac-D,L-seryl, N-Ac-D,L-threonyl,N-Ac-D,L-alanyl, 3-(1-naphthyl)-D,L-alanyl, 3-(2-naphthyl)-D,L-alanyl,3-(2,4,6-trimethylphenyl)-D,L-alanyl, and3-(4-trifluoromethylphenyl)-D,L-alanyl;

B is an amino acyl residue selected from the group consisting ofD-phenylalanyl, D-p-Cl-phenylalanyl, D-p-F-phenylalanyl,D-p-nitrophenylalanyl, 3-(3,4,5-trimethoxyphenyl)-D-alanyl,2,2-diphenylglycine, D-α-methyl-p-Cl-phenylalanine and3-(2,4,6-trimethylphenyl)-D-alanyl;

C is an amino acyl residue selected from the group consisting ofD-tryptophanyl, D-phenylalanyl, D-Me₅ phenylalanyl,3-(3-pyridyl)-D-alanyl, 3-(1-naphthyl)-D-alanyl, and3-(2-naphthyl)-D-alanyl;

D is an amino acyl residue selected from the group consisting ofL-seryl, and D-alanyl;

E is an amimo acyl residue selected from the group consisting ofL-phenylalanyl and L-tyrosyl;

F is an amino acyl residue selected from the group consisting of theradicals represented by the following structural formulas:

(a) ##STR3## wherein n is 1 to 5;

R₁ is alkyl of 1 to 12 carbon atoms, --NHR₃ wherein R₃ is hydrogen,alkyl of 1 to 12 carbon atoms, cycloalkyl, phenyl, benzyl, morpholino or--(CH₂)_(n) N(R₄)₂ wherein n is 1 to 5 and R₄ is lower alkyl;

R₂ is hydrogen or R₁ ; or R₁ and R₂ comprise a ring represented by thefollowing structural formulas: ##STR4## wherein n is 1 to 7; A ishydorgen, alkyl of 1 to 6 carbon atoms or cycloalkyl; and X is halo or Aor

(b) ##STR5## wherein R₅ is alkyl of 1 to 6 carbon atoms, benzyl,phenylethyl, cyclohexyl, cyclopentyl;

and R₆, R₇ and R₈ are hydrogen or methyl; and n is the integer 2-5; or

(c) a substituent of the formula ##STR6## wherein R₉ is hydrogen, alkylof 1 to 12 carbon atoms, phenyl or phenylloweralkyl;

G is an amino acyl residue selected from the group consisting ofL-leucyl, L-norleucyl and L-norvalyl;

H is D-alaninamide, D-leucinamide, glycinamide or --NHR₅ wherein R₅ islower alkyl or NHCONH₂ ; and the pharmaceutically acceptable saltsthereof.

The replacement of the L-histidyl residue which is at position 2 in LHRHwith one of the residues herein specified is a requirement to convertthe peptide to an LHRH antagonist. The replacement of the glycyl residueat position 6 in LHRH with one of the residues specified as F gives adramatic enhancement of the antagonist effect. The substitutionsdisclosed herein at positions 1, 2, 3, 4, 7 and 10 are further helpfulin enhancing the antagonist activity.

ABBREVIATIONS AND DEFINITIONS

As set forth above, and for convenience in describing this invention,the conventional abbreviations for the various common amino acids areused as generally accepted in the peptide art as recommended by theIUPAC-IUB Commission on Biochemical Nomenclature, Biochemistry, 11, 1726(1972). These represent L-amino acids, with the exception of the achiralamino acid glycine, and with the further exception of any unnatural ornatural amino acids which are achiral, or are otherwise designated asD-, and of those amino acids which are substituted herein into positions1, 2, 3, 4, 6, 7 and 10 for those normally found in LHRH. All peptidesequences mentioned herein are written according to the generallyaccepted convention whereby the N-terminal amino acid is on the left andthe C-terminal amino acid is on the right.

Certain other abbreviations will be useful in describing the invention.The present invention employs replacements by amino acids which do notoccur in nature. Particularly commonly employed among these are thefollowing:

    ______________________________________                                        Amino acid residue      Abbreviation                                          ______________________________________                                        3-(2-naphthyl)-D-alanyl D-Nal(2)                                              3-(p-fluorophenyl)-D-alanyl                                                                           D-p-F--Phe                                            3-(p-chlorophenyl)-D-alanyl                                                                           D-p-Cl--Phe                                           3-(2,3,4,5,6-pentamethylphenyl)-                                                                      D-Me                                                  D-alanyl                5Phe                                                  3-(2,4,6-trimethylphenyl)-D-alanyl                                                                    D-Tmp                                                 3-(3,4,5-trimethoxyphenyl)-D-alanyl                                                                   D-Tmo                                                 3-(4-(trifluoromethylphenyl)-D-alanyl                                                                 D-Ptf                                                 N,N'--guanido-dimethyl-D-                                                                             D-Dmh                                                 homoarginine                                                                  N,N'--guanido-diethyl-D-                                                                              D-Deh                                                 homoarginine                                                                  N,N'--guanido-dipropyl-D-                                                                             D-Dph                                                 homoarginine                                                                  N,N'--guanido-diisopropyl-D-                                                                          D-Dih                                                 homoarginine                                                                  N,N'--guanido-dihexyl-D-                                                                              D-Dhh                                                 homoarginine                                                                  N--guanido-isopropyl-D- D-Iph                                                 homoarginine                                                                  N--guanido-heptyl-D-homoarginine                                                                      D-Hha                                                 N--guanido-propyl-D-homoarginine                                                                      D-Prh                                                 N,N'--guanido-dicyclohexyl-D-                                                                         D-Dch                                                 homoarginine                                                                  N,N'--guanido-diisopropyl-D-                                                                          D-Dia                                                 arginine                                                                      N,N'--guanido-dicyclohexyl-D-                                                                         D-Dca                                                 arginine                                                                      N--guanido-(3-dimethylaminopropyl)-                                                                   D-Aph                                                 N'--guanido-ethyl-D-homoarginine                                              N--guanido-(3-dimethylaminopropyl)-                                                                   D-Apa                                                 N'--guanido-ethyl-D-arginine                                                  3-(3-piperdiyl)-D-alanine                                                                             D-3-Pia                                               3-(4-piperdiyl)-D-alanine                                                                             D-4-Pia                                               3-((N.sup.ε --methyl)piperid-4-yl)-D-                                                         D-Mpa                                                 alanine                                                                       3-((N.sup.ε --pentyl)piperid-4-yl)-D-                                                         D-Ppa                                                 alanine                                                                       3-((N.sup.ε --benzyl)piperid-4-yl)-D-                                                         D-Bpa                                                 alanine                                                                       ______________________________________                                    

As a further convenience, since the aminor acid sequence of LHRH hasbeen shown to be ##STR7## nona- and decapeptides in which the amino acidresidues at particular places in the sequence have been replaced byother amino acid residues or other moieties are abbreviated by showingthe nature of the substitution, superscribed by the location, followedby LHRH as the parent.

Thus, for example, the sequence, ##STR8## in which the Gly at position 6has been replaced by D-Dih and the His at position 2 has been replacedby D-p-F-Phe, is represented [D-p-F-Phe², D-Dih⁶ ]LHRH; and the sequence##STR9## is represented: [NAc-Pro¹, D-p-F-Phe², D-Dih⁶, Pro⁹ -NHEt]LHRH.

As used herein, the term "pharmaceutically acceptable salts" refers tosalts that retain the desired biological activity of the parent compoundand do not impart any undesired toxicological effects. Examples of suchsalts are (a) acid addition salts formed with inorganic acids, forexample hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoricacid, nitric acid and the like; and salts formed with organic acids suchas, for example, acetic acid, oxalic acid, tartaric acid, succinic acid,maleic acid, fumaric acid, gluconic acid, citric acid, malic acid,ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid,polyglutamic acid, naphthalenesulfonic acids, naphthalenedisulfonicacids, polygalacturonic acid; (b) salts with polyvalent metal cationssuch as zinc, calcium, bismuth, barium, magnesium, aluminum, copper,cobalt, nickel, cadmium, and the like; or with an organic cation formedfrom N,N'-dibenzylethylene-diamine or ethylenediamine; or (c)combinations, of (a) and (b), e.g., a zinc tannate salt and the like.

The term "lower alkyl" refers to a straight or branched chain saturatedhydrocarbon group having from 1 to 4 carbon atoms such as, for example,methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl andtert-butyl. "Alkyl of 1 to 6 carbon atoms" encompasses the samesubstituents as lower alkyl but in addition may have 5 or 6 carbon atomssuch as, for example, a n-pentyl, n-hexyl or other branched 5 or 6carbon membered moiety. "Alkyl of 1 to 12 carbon atoms" comprises aradical of 1 to 12 carbon atoms and hydrogen only as noted above, exceptthat the radical may have up to 12 carbon atoms. The term "cycloalkyl"refers to a cyclic saturated hydrocarbon group having from 3 to 6 carbonatoms, for example, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

For the purpose of this invention the abbreviation "alkylPro" refers tocis-5-alkyl-L-prolyl residue wherein alkyl is the same as "lower alkyl"defined above. More specifically "MePro" is cis-5-methyl-L-Prolyl,"EtPro" is cis-5-ethyl-L-Prolyl and "ButPro" is cis-5-n-butyl-L-Prolyl.

The abbreviation "N-Ac" refers specifically to the N-acetyl amino acidresidue in conformance with generally accepted nomenclature.

PREFERRED EMBODIMENTS OF THE COMPOUNDS

Compounds which are preferred embodiments of the present invention arethose wherein A is N-Ac-L-Pro, N-Ac-D-Ser, N-Ac-D-p-Cl-Phe,N-Ac-D-Nal(2); B is D-p-F-Phe or D-p-Cl-Phe; C is D-Trp, D-Nal(2) orD-Phe; D is Ser; E is Tyr; F is the compound of Formula II wherein n is3 or 4, R₁ is alkyl of 1 to 8 carbon atoms or cyclohexyl and R₂ ishydrogen, or a compound of Formula II wherein R₁ is --NHR₃ wherein R₃ ismethyl, ethyl, n-propyl, isopropyl, n-hexyl or cyclohexyl and R₂ is R₃or hydrogen, or F is a compound of Formula (IV) or (V) wherein R₉ ishydrogen, methyl, pentyl or benzyl; and H is D-AlaNH₂, GlyNH₂ or NHEt.

More preferred embodiments herein are:

A is N-Ac-L-Pro, N-Ac-D-Nal(2) or N-Ac-D-p-Cl-Phe, B is D-p-F-Phe orD-p-Cl-Phe, C is D-Nal(2) D-Trp or D-Phe, D is Ser, E is Tyr, F isD-Deh, D-Dph or D-Dhh and H is D-AlaNH₂ GlyNH₂ or NHEt;

N-Ac-L-Pro-D-p-F-Phe-D-Nal(2)-Ser-Tyr-F-Leu-Arg-Pro-GlyNH₂, wherein F isD-Deh, D-Dph, D-Dhh, D-Prh or D-Hha;

N-Ac-L-Pro-D-p-Cl-Phe-D-Trp-Ser-Tyr-F-Leu-Arg-Pro-NHEt, wherein F isD-Deh, D-Dph, D-Dhh, D-Prh or D-Hha;

N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-F-Leu-Arg-Pro-D-AlaNH₂, whereinF is D-Deh, D-Dph, D-Dhh, D-Prh or D-Hha;

N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-F-Leu-Arg-Pro-GlyNH₂, wherein Fis D-Deh, D-Dph, D-Dhh, D-Prh or D-Hha; and

N-Ac-D-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-F-Leu-Arg-Pro-GlyNH₂, wherein F isD-Deh, D-Dph, D-Dhh, D-Prh, D-Hha or propyl amidine.

The most particularly preferred embodiments are:

N-Ac-L-Pro-D-p-F-Phe-D-Trp-Ser-Tyr-D-Deh-Leu-Arg-Pro-NHEt;

N-Ac-L-Pro-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dph-Leu-Arg-Pro-NHEt;

N-Ac-L-Pro-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dhh-Leu-Arg-Pro-NHEt;

N-Ac-D-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Deh-Leu-Arg-Pro-NHEt;

N-Ac-D-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dph-Leu-Arg-Pro-NHEt;

N-Ac-D-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dhh-Leu-Arg-Pro-NHEt;

N-Ac-D-Nal(2)-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Deh-Leu-Arg-Pro-NHEt;

N-Ac-D-Nal(2)-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Dph-Leu-Arg-Pro-NHEt;

N-Ac-D-Nal(2)-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Dhh-Leu-Arg-Pro-NHEt;

N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Deh-Leu-Arg-Pro-NHEt;

N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Dph-Leu-Arg-Pro-NHEt;

N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Dhh-Leu-Arg-Pro-NHEt;

N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Deh-Leu-Arg-Pro-D-AlaNH₂ ;

N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Dph-Leu-Arg-Pro-D-AlaNH₂ ;and

N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Dhh-Leu-Arg-Pro-D-AlaNH₂.

In all of the above embodiments, the compound may also be prepared asthe corresponding pharmaceutically acceptable salt.

Assay Procedures

The compounds of this invention and, particularly, the salts thereof,exhibit surprisingly potent and long lasting LHRH antagonist activity.

Primary measures of potency are ability to inhibit ovulation in rats, asassayed by the procedure of Corbin, A. and Beattie, C. W., EndocrineRes. Commun., 2:1 (1975) and ability to inhibit LH release and ovulationin the rabbit, as per Phelps, C. P., et al, Endocrinology 100: (1977).

Other bioassays which are used for LHRH antagonists and for thecompounds of the present invention are:

(a) inhibition of LHRH induced FSH and LH release in the rat, in vivo;Vilchez-Martinez, J. A., et al, Endocrinology, 96: 1130 (1975); and,

(b) inhibition of LH and FSH release by dispersed anterior pituitarycell cultures as measured by radioimmuno assay. (Vale, W., et al,Endocrinology 91: 562 (1972).

Antagonist Effects and Utilities

The following utilities flow from the antagonist effect of the compoundsherein:

female contraception;

ovulation suppression or delay;

induction of parturition;

synchronization of ovulation;

estrus suppression;

growth promotion in female animals;

luteolysis, menses induction;

early, first trimester abortifacient;

therapy for endometriosis;

therapy for polycystic ovary syndrome (Stein-Leventhal);

therapy for benign prostatic hypertrophy;

male contraception;

therapy for diseases which result from excessive gonadal hormoneproduction in either sex;

functional castration in male food producing animals;

suppression of proestrous bloody discharge in dogs;

suppression of menopausal symptoms.

The aspect of the present invention which relates to particular uses forthe above-described compounds is concerned with these utilities, mostparticularly; inhibition of ovulation and treatment of endometriosis inthe female, and inhibition of spermatogenesis and treatment of prostatictumors in the male.

In the practice of the method of this invention an effective amount of acompound of the invention or a pharmaceutical composition containingsame is administered to the subject in need of, or desiring, suchtreatment. These compounds or compositions may be administered by any ofa variety of routes depending upon the specific end use, includingorally, parenterally (including subcutaneous, intramuscular andintravenous administration), vaginally (particularly for contraception),rectally, buccally (including sublingually), transdermally orintranasally. The most suitable route in any given case will depend uponthe use, particular active ingredient, the subject involved, and thejudgment of the medical practitioner. The compound or composition mayalso be administered by means of slow-release, depot or implantformulations as described more fully herein below.

In general for the uses herein above described, it is expedient toadminister the active ingredient in amounts between about 0.01 and 10mg/kg body weight per day, preferably between about 0.1 and 5.0 mg/kgbody weight per day. This administration may be accomplished by a singledaily administration, by distribution over several applications or byslow release in order to achieve the most effective results.

The exact dose and regimen for administration of these compounds andcompositions will necessarily be dependent upon the needs of theindividual subject being treated, the type of treatment, the degree ofaffliction or need and, of course, the judgment of the medicalpractitioner. In general, parenteral administration requires lowerdosage than other methods of administration which are more dependentupon absorption.

A further aspect of the present invention relates to pharmaceuticalcompositions containing as active ingredient a compound of the presentinvention which compositions comprise such compound in admixture with apharmaceutically acceptable, non-toxic carrier. As mentioned above, suchcompositions may be prepared for use for parenteral (subcutaneous,intramuscular or intravenous) administration particularly in the form ofliquid solutions or suspensions; for use in vaginal or rectaladministration particularly in semisolid forms such as creams andsuppositories; for oral or buccal administration particularly in theform of tablets or capsules; or intranasally particularly in the form ofpowders, nasal drops or aerosols.

The compositions may conveniently be administered in unit dosage formand may be prepared by any of the methods well-known in thepharmaceutical art, for example as described in Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 1970.Formulations for parenteral administration may contain as commonexcipients sterile water or saline, polyalkylene glycols such aspolyethylene glycol, oils of vegetable origin, hydrogenated naphthalenesand the like. Formulations for vaginal or rectal administration, e.g.suppositories, may contain as excipients, for example,polyalkyleneglycols, vaseline, cocoa butter, and the like. Formulationsfor inhalation administration may be solid and contain as excipients,for example, lactose or may be aqueous or oily solutions foradministration in the form of nasal drops. For buccal administrationtypical excipients include sugars, calcium stearate, magnesium stearate,pregelatinated starch, and the like.

It is particularly desirable to deliver the compounds of the presentinvention to the subject over prolonged periods of time, for example,for periods of one week to one year from a single administration.Various slow release, depot or implant dosage forms may be utilized. Forexample, a dosage form may contain a pharmaceutically acceptablenon-toxic salt of the compound which has a low degree of solubility inbody fluids, for example, (a) an acid addition salt with a polybasicacid such as phosphoric acid, sulfuric acid, citric acid, tartaric acid,tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenemono- or di-sulfonic acids, polygalacturonic acid, and the like; (b) asalt with a polyvalent metal cation such as zinc, calcium, bismuth,barium, magnesium, aluminum, copper, cobalt, nickel, cadmium and thelike, or with an organic cation formed from e.g.,N,N'-dibenzylethylenediamine or ethylenediamine; or (c) combinations of(a) and (b) e.g. a zinc tannate salt. Additionally, the compounds of thepresent invention or, preferably, a relatively insoluble salt such asthose just described, may be formulated in a gel, for example, analuminum monostearate gel with, e.g. sesame oil, suitable for injection.Particularly preferred salts are zinc salts, zinc tannate salts, pamoatesalts, and the like. Another type of slow release depot formulation forinjection would contain the compound or salt dispersed or encapsulatedin a slow degrading, non-toxic, non-antigenic polymer such as apolylactic acid/polyglycolic acid polymer for example as described inU.S. Pat. No. 3,773,919. The compounds or, preferably, relativelyinsoluble salts such as those described above may also be formulated incholesterol matrix pellets, particularly for use in animals. Additionalslow release, depot or implant formulations, e.g. liposomes, are wellknown in the literature. See, for example, Sustained and ControlledRelease Drug Delivery Systems, J. R. Robinson ed., Marcel Dekker, Inc.,New York, 1978. Particular reference with respect to LHRH type compoundsmay be found, for example, in U.S. Pat. No. 4,010,125.

Synthesis of the Peptides

The polypeptides of the present invention may be synthesized by anytechniques that are known to those skilled in the peptide art. Anexcellent summary of the many techniques so available may be found in J.M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, W. H. FreemanCo., San Francisco, 1969, and J. Meienhofer, Hormonal Proteins andPeptides, Vol. 2, p. 46., Academic Press (New York), 1973 for solidphase peptide synthesis and E. Schroder and K. Lubke, The Peptides, Vol.1, Academic Press (New York), 1965 for classical solution synthesis.

In general, these methods comprise the sequential addition of one ormore amino acids or suitably protected amino acids to a growing peptidechain. Normally, either the amino or carboxyl group of the first aminoacid is protected by a suitable protecting group. The protected orderivatized amino acid can then be either attached to an inert solidsupport or utilized in solution by adding the next amino acid in thesequence having the complimentary (amino or carboxyl) group suitablyprotected, under conditions suitable for forming the amide linkage. Theprotecting group is then removed from this newly added amino acidresidue and the next amino acid (suitably protected) is then added, andso forth. After all the desired amino acids have been linked in theproper sequence, any remaining protecting groups (and any solid support)are removed sequentially or concurrently, to afford the finalpolypeptide. By simple modification of this general procedure, it ispossible to add more than one amino acid at a time to a growing chain,for example, by coupling (under conditions which do not racemize chiralcenters) a protected tripeptide with a properly protected dipeptide toform, after deprotection, a pentapeptide.

PREFERRED EMBODIMENTS OF SYNTHESIS

A particularly preferred method of preparing compounds of the presentinvention involves solid phase peptide synthesis.

In this particularly preferred method the ]-amino function of the aminoacids is protected by an acid or base sensitive group. Such protectinggroups should have the properties of being stable to the conditions ofpeptide linkage formation, while being readily removable withoutdestruction of the growing peptide chain or racemization of any of thechiral centers contained therein. Suitable protecting groups aret-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz),biphenylisopropyloxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl,],]-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl,2-cyano-t-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl and the like,especially t-butyloxycarbonyl (Boc).

Particularly preferred side chain protecting groups are, forarginine:nitro, p-toluenesulfonyl, 4-methoxybenzenesulfonyl, Cbz, Bocand adamantyloxycarbonyl; for tyrosine:benzyl, o-bromobenzyloxycarbonyl,2,6-dichlorobenzyl, isopropyl, cyclohexyl, cyclopentyl and acetyl; forserine:benzyl and tetrahydropyranyl; for histidine:benzyl,p-toluenesulfonyl and 2,4-dinitrophenyl.

The C-terminal amino acid is attached to a suitable solid support.Suitable solid supports useful for the above synthesis are thosematerials which are inert to the reagents and reaction conditions of thestepwise condensation-deprotection reactions, as well as being insolublein the media used. Suitable solid supports arechloromethylpolystyrene-divinylbenzene polymer,hydroxymethyl-polystyrene-divinylbenzene polymer, and the like,especially chloromethyl-polystyrene-1% divinylbenzene polymer. For thespecial case where the C-terminus of the compound will be glycinamide, aparticularly useful support is thebenzhydrylamino-polystyrene-divinylbenzene polymer described by P.Rivaille, et al, Helv. Chim. Acta., 54, 2772 (1971). The attachment tothe chloromethyl polystyrene-divinylbenzene type of resin is made bymeans of the reaction of the N.sup.α -protected amino acid, especiallythe Boc-amino acid, as its cesium, tetramethylammonium,triethylammonium, 1,5-diazabicyclo[5.4.0]undec-5-ene, or similar salt inethanol, acetonitrile, N,N-dimethylformamide (DMF), and the like,especially the cesium salt in DMF, with the chloromethyl resin at anelevated temperature, for example between about 40° and 60° C.,preferably about 50° C., for from about 12 to 48 hours, preferably about24 hours. The N.sup.α -Bocamino acid is attached to the benzhydrylamineresin by means of an N,N'-dicyclohexylcarbodiimide(DCC)/1-hydroxybenzotriazole (HBT) mediated coupling for from about 2 toabout 24 hours, preferably about 12 hours at a temperature of betweenabout 10° and 50° C., preferably 25° C. in a solvent such asdichloromethane or DMF, preferably dichlormethane. The coupling ofsuccessive protected amino acids can be carried out in an automaticpolypeptide synthesizer as is well known in the art. The removal of theN.sup.α -protecting groups may be performed in the presence of, forexample, a solution of trifluoroacetic acid in methylene chloride,hydrogen chloride in dioxane, hydrogen chloride in acetic acid, or otherstrong acid solution, preferably 50% trifluoroacetic acid indichloromethane at about ambient temperature. Each protected amino acidis preferably introduced in approximately 2.5 molar excess and thecoupling may be carried out in dichloromethane, dichloromethane/DMFmixtures, DMF and the like, especially in methylene chloride at aboutambient temperature. The coupling agent is normally DCC indichloromethane but may be N,N'-di-iso-propylcarbodiimide (DIC) or othercarbodiimide either alone or in the presence of HBT,N-hydroxysuccin-imide, other N-hydroxyimides or oximes. Alternately,protected amino acid active esters (e.g. p-nitrophenyl,pentafluorophenyl and the like) or symmetrical anhydrides may be used.

At the end of the solid phase synthesis the fully protected polypeptideis removed from the resin. When the linkage to the resin support is ofthe benzyl ester type, cleavage is by means of aminolysis with analkylamine or fluoroalkylamine for peptides with a proline C-terminus,or by aminolysis with, for example, ammonia/methanol or ammonia/ethanolfor peptides with a glycine C-terminus at a temperature between about10° and 50° C., preferably about 25° C., for between about 12 and 24hours preferably about 18 hours. Alternatively, the peptide may beremoved from the resin by transesterification, e.g., with methanol,followed by aminolysis. The protected peptide may be purified at thispoint by silica gel chromatography. The removal of the side chainprotecting groups from the polypeptide is performed by treating theaminolysis product with, for example, anhydrous liquid hydrogen fluoridein the presence of anisole or other carbonium scavenger, treatment withhydrogen fluoride/pyridine complex, treatment withtris(trifluoroacetyl)boron and trifluoroacetic acid, by reduction withhydrogen and palladium on carbon or polyvinylpyrrolidone, or byreduction with sodium in liquid ammonia, preferably with liquid hydrogenfluoride, and anisole at a temperature between about -10° and +10° C.,preferably about 0° C., for between about 15 minutes and 1 hour,preferably about 30 minutes. For the glycine terminal peptides on thebenzyhydrylamine resins, the resin cleavage and deprotection steps maybe combined in a single step utilizing liquid hydrogen fluoride andanisole as described above. The fully deprotected polypeptide is thenpurified by a sequence of chromatographic steps employing any or all ofthe following types: ion exchange on a weakly basic resin in the acetateform; hydrophobic adsorption chromatography on underivatizedpolystyrene-divinylbenzene (for example Amberlite XAD); silica geladsorption chromatography; ion exchange chromatography oncarboxymethylcellulose; partition chromatography, e.g., on SephadexG-25, or countercurrent distribution; high performance liquidchromatography (HPLC), especially reverse phase HPLC on octyl- oroctadecylsilyl-silica bonded phase column packing.

If a racemic amino acid is used in the 1, 2, 3 or 6 position, thediastereomeric nonapeptide or decapeptide final products are separated,and the desired peptide containing a D-amino acid in the appropriateposition is isolated and purified, preferably during the above-describedchromatographic process.

The preparation of peptides having C-terminal azaglycine amides ispreferably done using classical peptide solution synthesis using knownpeptide intermediates. This is described in more detail in Example 3.

Thus, in another aspect the present invention relates to a method forpreparing compounds of the invention and of the pharmaceuticallyacceptable salts thereof which process comprises:

removing protecting groups and, optionally, covalently bound solidsupport from a protected polypeptide to afford a compound of Formula (I)or a salt thereof, and optionally

(a) converting a compound of Formula (I) to a pharmaceuticallyacceptable salt, or

(b) converting a salt of a compound of Formula (I) to a pharmaceuticallyacceptable salt, or

(c) decomposing a salt of a compound of Formula (I) to a freepolypeptide of Formula (I).

The following examples are given to enable those skilled in the art tomore fully understand and practice the present invention. They shouldnot be construed as a limitation upon the scope of the invention, butmerely as being illustrative and representative thereof.

PREPARATION A N-ACETYL 3-(2-NAPHTHYL)-D,L-ALANINATE

The preparation of 3-(2-naphthyl)-D,L-alanine is carried out accordingto the prodecure set out in U.S. Pat. No. 4,341,767.

Preparation of N acetyl 3-(2-naphthyl)-D,L-alinine, its conversion tomethyl N acetyl 3-(2-naphthyl)-D,L-alinine, and separation of the Disomer is carried out by the procedure disclosed in U.S. Pat. No.4,341,767.

PREPARATION B A mixture of 5.24 g of benzyl N.sub.α-benzyloxy-carbonyl-D-lysinate toluenesulfonate (B. Bezus and L.Zeravus, J. Am. Chem. Soc. 83, 719 (1961)) and 1.72 ml ofdiisopropylethylamine in 60 ml of dioxane is treated with 1.89 g ofN,N'-diisopropylcarbodiimide. The reaction mixture is stirred at 100° C.for 6 hours, cooled to room temperature and concentrated to a solid. Thesolid is suspended in 20 ml of warm DMF, filtered to removeN,N'-diisopropylurea and the filtrate concentrated to a solid. BenzylN.sub.α -benzyloxycarbonyl-N,N'-guanido-diisopropyl-D-homoargininatetoluenesulfonate is obtained as a white solid by crystallization frommethanol/ethyl acetate [α]_(D) -7.26° (C 0.3, MeOH). Similarly, by usingthe above procedure, but substituting:

N,N'-dicyclohexylcarbodiimide;

N,N'-di-n-hexylcarbodiimide;

N,N'-diethylcarbodiimide;

N,N'-di-n-propylcarbodiimide;

N-i-propylcarbodiimide;

N-propylcarbodiimide;

N,N'-di-n-butylcarbodiimide;

N,N'-dimethylcarbodiimide;

N,N'-di-i-butylcarbodiimide;

N,N'-di-n-pentylcarbodiimide;

N,N'-di-i-pentylcarbodiimide;

N,N'-diphenylcarbodiimide;

N,N'-ditolylcarbodiimide; or

1-(3-dimethylaminopropyl)-3-ethylcarbodiimide-HCl

and the like, there are obtained: benzyl N.sup.α-benzyloxycarbonyl-N,N'-guanido-dicyclohexyl-D-homoargininate, [α]_(D)8.07° (C 0.9 MeOH);

benzyl N.sup.α -benzyloxycarbonyl-N,N'-guanido-diethyl-D-homoargininate;

benzyl N.sup.α-benzyloxycarbonyl-N,N'-guanido-di-n-propyl-D-homoargininate [α]_(D)8.07° (C 0.9 MeOH);

benzyl N.sup.α -benzyloxycarbonyl-NN'-guanido-n-propyl-D-homoargininate;

benzyl N.sup.α-benzyloxycarbonyl-N,N'-guanido-di-n-butyl-D-homoargininate;

benzyl N.sup.α-benzyloxycarbonyl-N,N'-guanido-di-i-butyl-D-homoargininate;

benzyl N.sup.α-benzyloxycarbonyl-N,N'-guanido-di-n-pentyl-D-homoargininate;

benzyl N.sup.α-benzyloxycarbonyl-N,N'-guanido-di-phenyl-D-homoargininate;

benzyl N.sup.α-benzyloxycarbonyl-N,N'-guanido-dimethyl-D-homoargininate;

benzyl N.sup.α-benzyloxycarbonyl-N,N'-guanido-di-n-hexyl-D-homoargininate;

benzyl N.sup.α-benzyloxycarbonyl-N,N'-guanido-di-isopropyl-D-argininate, [α]_(D)-10.5° (C 0.5, MeOH);

benzyl N.sup.α -benzyloxycarbonyl,N-guanido-(3-dimethylaminopropyl)-N'-guanido-ethyl-D-homoargininate astheir benzenesulfonate salts. Similiarly, by substituting benzyl N.sup.α-benzyloxycarbonyl-D-ornithinate for the D-lysinate there may beobtained the corresponding arginine analogs as their toluenesulfonatesalts.

PREPARATION C Benzyl N.sup.α -benzyloxycarbonyl-N^(G),N^(G')-etheno-D-homoargininate

To a mixture of 15 ml of toluene and 15 ml of t-BuOH was added 2.71 g ofbenzyl N.sup.α -benzyloxycarbonyl-D-lysinate and 1.46 g of2-methylthioimidazoline.HI (available from Aldrich). The pH of themixture was brought to ˜8 by the addition of diisopropylethylamine andthe solution heated under reflux for 24 hours.

The solution was concentrated in vacuo and the residue was loaded on asilica gel column (250 g). The column was eluted with a gradient fromCH₂ Cl₂ /MeOH (19:1) to CH₂ Cl₂ /MeoH (7:3). The fractions containingproduct were detected by TLC, pooled, and concentrated to dryness, 2.9 gof white foam.

A 2 g portion of the above-named product was dissolved in 50 ml of EtOHcontaining 0.8 g of 10% Pd/C. The solution was stirred under H₂ for 8hours. The mixture was filtered on celite and the filtrate wasconcentrated to dryness to give N^(G),N^(G') -etheno-D-homoarginine as awhite foam, 1.2 g.

PREPARATION D

This Preparation illustrates the preparation of N.sup.α -t-butyloxycarbonyl derivatives of N,N'-guanido-disubstituted-D-homoarginines fromtheir toluenesulfonate precursors.

A mixture of N,N'-guanido-diisopropyl-D-homoargininate toluenesulfonate(3.25 g) and 100 mg of 10% Pd/C in 50 ml of glacial acetic acid istreated with hydrogen gas at atmospheric pressure for 4 hours. Thecatalyst is filtered on celite and the filtrate is concentrated to asolid, N,N'-guanido-diisopropyl-D-homoarginine toluenesulfonate. Asolution of this compound (2.13 g) in 60 ml of 50% dioxane/water istreated with 10 ml of 1N sodium hydroxide and 0.4 g of magnesium oxide.This mixture is then treated with 1.1 g of di-t-butyldicarbonate andstirred at room temperature for 2 hours. The magnesium salt is filteredand the filtrate is concentrated under vacuum. The basic solution iswashed with ethanol, then brought to pH 2.5 with sodium sulfate. Theacidic aqueous solution is extracted with ethylacetate which is driedover magnesium sulfate. The drying agent is filtered and the filtrate isconcentrated. Crystallization from ethyl acetate/hexane affords N.sup.α-t-butyloxycarbonyl,N,N'-guanido-diisopropyl-D-homoargininetoluenesulfonate.

Proceeding in a similiar manner, but substituting the appropriatetoluenesulfonate precursors, other N.sup.α-t-butyloxycarbonyl-N,N'-guanido-disubstituted-D-homoarginine orD-arginine compounds may be prepared.

PREPARATION E

N.sub.α -t-butyloxycarbonyl-3-(4'-(1'-propylpiperidyl))-D-alanine

A 4.6 g portion of sodium metal was added to 400 ml of absolute ethanoland heated. To the resultant solution of sodium ethoxide was added 21.7g of diethyl acetamidomalonate and 16.4 g of 4-picolyl chloridehydrochloride (Aldrich Chem. Co.). The reactin mixture was heated to100° C. for 4 hours, cooled, filtered and concentrated in vacuo. Themixture was loaded on a silica gel column in methylene chloride/methanol(19:1) and eluted with the same mixture. The product was located as afast running UV positive spot by TLC on silica gel in methylenechloride/methanol (19:1). Combined fractions were concentrated toprovide the product.

The product from the foregoing paragraph was dissolved in 200 ml ofethanol and treated with a solution of 2.72 g of sodium hydroxide in 40ml of water at 50° C. for 6 hours. The solution was acidified with 12 mlof 6N HCl, concentrated to dryness and taken up in 200 ml of dioxane.The suspension was filtered and the filtrate heated at reflux for 2hours. The solution was cooled and concentrated to dryness to yieldethyl N.sup.α -acetyl-3-(4-pyridyl)-D,L-alanine as a white solid.

A portion of this N-acetyl ester was resolved by treatment with 200 mlof the enzyme subtilisin Carlsberg (Sigma Chem. Co., protease VIII) in amixture of 300 ml of dimethyl sulfoxide and 400 ml of 0.01M KCl (pH7.2). The pH was maintained by addition of 1N NaOH on a pH stat. After a6 hour period, the resolution was complete. The solution was dilutedwith 400 ml of water and extracted with 4×750 ml of ethyl acetate. Theorganic layers were combined and dried over magnesium sulfate andconcentrated to yield ethyl N.sup.α -acetyl-3-(4-pyridyl)-D-alaninate asan oil.

The oil was reacted with 1.22 g of n-propyl bromide in 50 ml of ethanolafter which the solution was concentrated to dryness to yield ethylN.sup.α -acetyl-3-(1-propyl-pyridinium-4-yl)-D-alininate bromide as awhite hygroscopic solid.

This white solid was dissolved in 200 ml of ethanol and was reducedunder an atmosphere of hydrogen gas using 100 mg of 10% Pd/C as acatalyst. After an 18 hour reduction period, the catalyst was filteredout and the solutin concentrated to yield ethyl N.sup.α-acetyl-3-(4'-(1'-propylpiperidyl))-D-alininate as a tan solid. The freeacid was prepared by refluxing the ethyl ester in 100 ml of 6N HCl for 4hours to yield 3-(4'-(1'-propylpiperidyl))-D-alanine as a white solid.

The free acid was dissolved in 100 ml of dioxane/water (1:1) and treatedwith 2 g of di-t-butyldicarbonate. The pH was maintained at 9 byaddition of 1N NaOH on a pH stat. After 2 hours the reaction muxture wasconcentrated in vacuo, washed wtih 100 ml of ethyl ether and the aqueouslaye was loaded on an Amberlite XAD-2 hydrophobic resin. The column waseluted with 250 ml of water followed by 250 ml of 50% ethanol/ater. Theethanol eluate was pooled and concentrated to dryness to yield N.sup.α-t-butyloxy-carbonyl-3-(4'-(1'-propylpiperidyl))-D-alanine as a whitesolid.

Proceeding in similiar manner, but substituting 3-picolyl chloridehydrochloride for 4-picolyl chloride hydrochloride, there is preparedN.sup.α -t-butyloxy-carbonyl-3-(3'-(1'-propylpiperidyl))-D-alanine.

PREPARATION F

Cis-5-alkylproline compounds may be prepared by the following method:

To a 200-ml round-bottomed flask is added(S)-3-(benzyloxycarbonyl)-5-oxo-4-oxazolidinepropionic acid and 63 ml ofanhydrous benzene. To this solution is added 13.9 g of phosphoruspentachloride at 0° C. The reaction mixture is stirred at 0° C. for 1 hrduring which time all of the phosphorus pentachloride dissolves. Thebenzene solvent is removed under vacuum and coevaporation with two 25 mlsamples of dry benzene, and the residue dried under vacuum to give alight solid. The light solid is suspended in 30 ml ofhexamethylphosphoramide and 9.4 ml of tetramethyltin and 40 mg of PhCH₂Pd(PPh₃)₂ Cl is added. The reaction mixture is heated at 65° C. for 4hours. An additional 2 ml of tetramethyltin is added at the end of thatperiod and the reaction mixture is stirred over night at roomtemperature.) After dilution with water and extraction with ethylacetate, the ethyl acetate layer is washed with water, 5% sodiumbicarbonate, water, 5% sodium bisulfate, water, and saturated sodiumchloride and dried over anhydrous magnesium sulfate. The solution isfiltered and concentrated to give 16 g of a yellow oil, which is passedthrough a silica gel column using ethyl acetate/hexane(4/6) as eluent.Concentration of the appropriate fractions gives 15 g of a light yellowoil which is recrystallized from ethyl acetate-hexane to produce 14.3 gof (S)-3-(benzyloxycarbonyl)-4-(3-oxobutyl)-5-oxazolidinone as a whitesolid (74% yield), having a mp of 64°-65° C., [α]_(D) ²⁵ =+102° (c=1.1,CH₂ Cl₂).

Anal: Calcd. for C₁₈ H₁₇ NO₅ : C, 61.85; H, 5.84;N, 4.81. Found: C,61.54; H, 5.89;N, 4.84.

By repeating the above procedure in a similar manner, and, by replacingthe tetramethyltin with a stoichiometrically equivalent of theappropriate tetraalkyltinthe following compounds are prepared:

(a) With tetraethyltin:

(S)-3-(Benzyloxycarbonyl)-4-(3-oxopentyl)-5-oxazolidinone having a mp of45°-46° C.;

[α]_(D) ²⁵ =82.5° (c 0.7, CHhd 3OH).

Anal: Calcd. for C₁₆ H₁₉ NO₅ (305.336): C,62.94; H,6.37;N,4.59. Found:C,63.02; H,6.15;N,4.48.

(b) With tetrabutyltin:

(S)-3-(Benzyloxycarbonyl)-4-(3-oxoheptyl)-5-oxazolidinone as an oil;[α]_(D) ²⁵ 67.9° (c 0.12, CH₃ OH).

Anal: Calcd. for C₁₈ H₂₃ NO₅ EtOAc(421.494): C,62.69; H,7.41;N,3.32.Found: C,62.50; H,7.29;N,3.39.

Ten grams of the(S)-3-(benzyloxycarbonyl)-4-(3-oxobutyl)-5-oxazolidinone from Example 4is dissolved in 480 ml of distilled tetrahydrofuran, followed by 160 mlof ammonia at 0° C. The reaction mixture is stirred at 0° for 5 hours,then at ambient temperature overnight. After stripping under vacuum todryness, the reaction mixture yields a white solid which isrecrystallized from hot ethyl acetate to give 8.8 g of(S)-2-(benzyloxycarbonylamino)-5-oxo-hexanamide as a white solid (82%yield), mp 142°-144°; [α]_(D) ²⁵ =-4.0° (c 0.4, CH₃ OH).

Anal.: Calcd. for C₇ H₉ NO₂ : C, 60.4; H, 6.4;N, 10.0. Found: C, 60.44;H, 6.53;N, 10.05.

By repeating the above procedure in a similar manner and substituting astoichiometrically equivalent of the corresponding intermediates fromthe second previous paragraph, the following compounds are prepared :

(S)-2-Benzyloxycarbonylamino-5-oxo-heptanamide having a mp of 133°-135°C.; [α]_(D) ²⁵ -4.17° (c 0.8, CH₃ OH).

Anal: Calcd. for C₁₅ H₂₀ N₂ O₄ (292.341): C,61.63; H,6.90;N,9.58. Found:C,61.51; H,6.75;N,9.16.

(S)-2-Benzyloxycarbonylamino-5-oxo-nonamide having a mp of 162°-163°C.;[α]_(D) ²⁵ -4.32° (c 0.6, CH₃ OH).

Anal: Calcd. for C₁₇ H₂₄ N₂ O₄ (320.395): C,63.73; H,7.55;N,8.74.C,63.62; H,7.56;N,8.82.

To a solution of 2.8 g of (S)-2-benzyloxycarbonylamino)-5-oxohexanamidefrom the preceeding paragraph in a mixture of 60 ml of methanol and 7.5ml of glacial acetic acid is added, under nitrogen, 1.5 g of palladiumdiacetate. This reaction mixture is hydrogenated under atmosphericpressure for 4 hrs, at which time a thin layer chromagraphic analysisshows the reaction had gone to completion. The reaction mixture is thenfiltered through Celite and washed with methanol. The reaction mixtureand washings are concentrated to dryness to give 1.7 g of a yellow oil,which is treated with 1 ml of a mixture of hydrochloric acid and ethylacetate to produce the hydrochloride salt. This oil is triturated withmethanol/ethyl ether to produce 1.3 g of a yellow solid; mp 174°-176°C.;[α]_(D) ²⁵ =-33° (c 0.96, CH₃ OH). The yellow solid of(S)-cis-5-methylprolinamide (as the hydrochloride salt) is passedthrough a Bio-Rex 70 column (a weakly acid carboxylic acid ion-exchangeresin) with elution first with 300-ml of water, followed by 1% solutionof ammonium hydroxide. Concentration of the appropriate fractions givesa 0.9 g of a yellow solid, which is recrystallized from methylenechloride to produce 0.64 g of (S)-cis-S-methylprolinamide as a yellowsolid (50% yield); mp 55°-56° C.

By repeating the above procedure in a similar fashion, and substitutinga stoichiometrically equivalent of the corresponding intermediate, thefollowing compounds are prepared after reduction:

(S)-cis-5-ethylprolinamide, mp 63°-65° C.; and

(S)-cis-5-butylyprolinamide, mp 74°-75° C.

PREPARATION G

4.9 g of Boc-glycine was dissolved in a mixture of 50 ml. ethanol and 50ml. distilled water. The pH of the solution was brought to 7 withaqueous cesium bicarbonate. The solvent was then removed under vacuum.

After 18 hours of drying under high vacuum, the residue was dissolved in150 ml. dry DMF. 25 g chloromethylated polystyrene --1% divinylbenzene(Merrifield) resin (corresponding to 25 mmole chloride) was added. Themixture was shaken at 50° C. for 24 hours, filtered, and the resin wasthen washed sequentially with DMF, water, and ethanol. The resin wasdried under vacuum for 3 days to yield 28.34 g of Boc-Gly-O-Resin.

EXAMPLE 1

In the reaction vessel of a Beckman 990 Peptide Synthesizer was placed7.29 g. (5.5 mmol.) of Boc-Gly-O-Resin prepared by the reaction ofequimolar ratios of the dry cesium salt of Boc-GlyOH withchloromethylpolystyrene/1% divinylbenzene. Amino acids were addedsequentially to this resin by means of a synthesis program, as follows:

    ______________________________________                                        Step 1  CH.sub.2 Cl.sub.2 wash                                                                          1 time   1.5 min                                    2       50% CF.sub.3 CO.sub.2 H/CH.sub.2 Cl.sub.2 -                                                     1 time   1.5 min                                            deprotection                                                          3       50% CF.sub.3 CO.sub.2 H/CH.sub.2 Cl.sub.2 -                                                     1 time   30 min                                             deprotection                                                          4       CH.sub.2 Cl.sub.2 wash                                                                          3 times  1.5 min                                    5       10% triethylamine/CH.sub.2 Cl.sub.2                                                             2 times  1.5 min                                    6       CH.sub.2 Cl.sub.2 wash                                                                          3 times  1.5 min                                    7       N.sup.α --Boc--amino acid                                                                 1 time   add                                                solution                                                              8       N,N'--dicyclohexylcarbo-                                                                        1 time   add                                                diimide solution                                                      9       CH.sub.2 Cl.sub.2 rinse and hold-                                                               1 time   coupling                                                                      reaction                                           coupling                   2 hr                                       10      CH.sub.2 Cl.sub.2 - rinse add                                                                   1 time   1.5 min                                    11      CH.sub.2 Cl.sub.2 wash                                                                          3 times  1.5 min                                    12      ethanol wash      3 times  1.5 min                                    13      CH.sub.2 Cl.sub.2 wash                                                                          3 times  1.5 min                                    ______________________________________                                    

Steps 1-13 complete a coupling cycle for one amino acid and completenessof the reaction is checked by the ninhydrin method of E. Kaiser, et al.,Anal. Biochem., 34, 595 (1970).

The resin was coupled sequentially with a 2.0 to 2.5 molar excess ofeach protected amino acid and DCC. Thus, the resin was treated duringsuccessive coupling cycles with

3.01 g. Boc-Pro-OH,

5.99 g. Boc-ArG (Iosyl)-OH,

3.49 g. Boc-Leu-OH.H₂ O

At this point, the resulting tetrapeptide resin was divided into smallerbatches. A 1.00 g. batch was carried forward by further coupling insuccessive cycles with

0.456 g. Boc-D-Dia-OH toluenesulfonate,

0.44 g. Boc-Tyr(2,6-dichlorobenzyl)-OH,

0.375 g. Boc-Ser(Benzyl)-OH,

0.315 g. Boc-D-Nal(2)-OH,

0.35 g. Boc-D-p-F-Phe-OH,

0.275 g. Boc-L-Proline, and

2.5 ml. acetic anhydride.

The resin was removed from the reaction vessel, washed with CH₂ Cl₂, anddried in vacuo to yield 1.66 g. of protected polypeptide resin. Theprotected peptide was removed from the resin by treatment at roomtemperature for 24 hours with 50 ml. of methanol saturated at 0° C. withammonia. The resin beads were filtered and washed sequentially withmethanol and DMF. Solvent was removed from the filtrate under vacuum toyield the protected peptide as 0.9 g. of yellow oil.

The protecting groups were removed by treatment with 10 ml. anhydrousliquid HF in the presence of 1 ml. of anisole (scavenger) in a Kel-Freaction vessel at 0° C. for 30 minutes. The HF was evaporated undervacuum and the residue ofN-Ac-L-Pro-D-p-Cl-Phe-D-Nal(2)-Ser-Tyr-D-Dih-Leu-Arg-Pro-GlyNH₂, as itsHF salt, was washed with ether. The residue was then extracted withglacial acetic acid. The acetic acid extract was lyophilized to yield0.5 g. of crude material.

The crude material was converted to the acetate salt by passage in waterthrough a column of AG3X (a weakly basic tertiary amine resin) which hadbeen converted to the acetate form. Lyophilization of the eluate yielded0.5 g. of the crude peptide acetate salt as a white solid.

The crude peptide was purified by high performance liquid chromatographyon a 2.5×100 cm. column of Licroprep Rp-18 (25-40 micron) equilibratedto the running buffer 35% CH₃ CN/65%H₂ O (0.03 M in NH₄ OAc, pH 4.5).The major UV absorbing (280 nm) peak eluting at approximately 3 columnvolumes was collected, concentrated to dryness, and lyophilized 3 timesfrom distilled water to yield 75 mg of pureN-Ac-L-Pro-D-p-F-Phe-D-Nal(2)-Ser-Tyr-D-Dia-Leu-Arg-Pro-GlyNH₂, [α]_(D)²⁵ =-15.4° (C 0.5, HOAc).

Proceeding in a similiar manner but substituting the appropriate A, B,C, D, E, G or F amino acid for those recited, there are prepared thecorresponding GlyNH₂ decapeptides exemplified below.

N-Ac-L-Pro-D-p-F-Phe-D-Nal(2)-Ser-Tyr-D-Dih-Leu-Arg-Pro-GlyNH₂,

N-Ac-L-Pro-D-p-F-Phe-D-Nal(2)-Ser-Tyr-D-Iph-Leu-Arg-Pro-GlyNH₂,

N-Ac-D-Nal(2)-D-p-F-Phe-D-Nal(2)-Ser-Tyr-D-Iph-Leu-Arg-Pro-GlyNH₂,

N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Phe-Ser-Tyr-D-Iph-Leu-Arg-GlyNH₂,

N-Ac-L-Pro-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dih-Leu-Arg-Pro-GlyNH₂,

N-Ac-L-Pro-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dmh-Leu-Arg-Pro-GlyNH₂,

N-Ac-L-Pro-D-p-F-Phe-D-Trp-Ser-Tyr-D-Deh-Leu-Arg-Pro-GlyNH₂,

N-Ac-L-Pro-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dph-Leu-Arg-Pro-GlyNH₂,

N-Ac-L-Pro-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dhh-Leu-Arg-Pro-GlyNH₂,

N-Ac-L-Pro-D-p-F-Phe-D-Trp-Ser-Tyr-D-Prh-Leu-Arg-Pro-GlyNH₂,

N-Ac-L-Pro-D-p-F-Phe-D-Trp-Ser-Tyr-D-Hha-Leu-Arg-Pro-GlyNH₂,

N-Ac-L-Pro-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dpa-Leu-Arg-Pro-GlyNH₂,

N-Ac-L-Pro-D-p-F-Phe-D-Trp-Ser-Tyr-D-Mpa-Leu-Arg-Pro-GlyNH₂,

N-Ac-L-Pro-D-p-F-Phe-D-Trp-Ser-Tyr-D-Pia-Leu-Arg-Pro-GlyNH₂,

N-Ac-L-Pro-D-p-F-Phe-D-Trp-Ser-Tyr-D-Ppa-Leu-Arg-Pro-GlyNH₂,

N-Ac-L-Pro-D-p-F-Phe-D-Trp-Ser-Tyr-D-Bpa-Leu-Arg-Pro-GlyNH₂,

N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Dmh-Leu-Arg-Pro-GlyNH₂,

N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Deh-Leu-Arg-Pro-GlyNH₂,

N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Dph-Leu-Arg-Pro-GlyNH₂,

N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Dih-Leu-Arg-Pro-GlyNH₂,

N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Dhh-Leu-Arg-Pro-GlyNH₂,

N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Prh-Leu-Arg-Pro-GlyNH₂,

N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Hha-Leu-Arg-Pro-GlyNH₂,

N-Ac-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dmh-Leu-Arg-Pro-GlyNH₂,

N-Ac-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Deh-Leu-Arg-Pro-GlyNH₂,

N-Ac-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dph-Leu-Arg-Pro-GlyNH₂,

N-Ac-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dih-Leu-Arg-Pro-GlyNH₂,N-Ac-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dhh-Leu-Arg-Pro-GlyNH₂,N-Ac-Nal(2)-D-p-F-Phe-D-Nal(2)-Ser-Tyr-D-Dph-Leu-Arg-Pro-GlyNH₂,N-Ac-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dhh-Leu-Arg-Pro-GlyNH₂,N-Ac-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Prh-Leu-Arg-Pro-GlyNH₂,N-Ac-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Hha-Leu-Arg-Pro-GlyNH₂,N-Ac-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-PropylAmidine-Leu-Arg-Pro-GlyNH₂,N-Ac-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Mpa-Leu-Arg-Pro-GlyNH₂,N-Ac-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Pia-Leu-Arg-Pro-GlyNH₂,N-Ac-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Ppa-Leu-Arg-Pro-GlyNH₂,N-Ac-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Bpa-Leu-Arg-Pro-GlyNH₂,N-Ac-Nal(2)-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Dph-Leu-Arg-Pro-GlyNH₂,N-Ac-Nal(2)-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Dmh-Leu-Arg-Pro-GlyNH₂,N-Ac-Nal(2)-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Deh-Leu-Arg-Pro-GlyNH₂, andN-Ac-Nal(2)-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Dhh-Leu-Arg-Pro-GlyNH₂.

In a similar manner, by replacing the Boc-Gly-O-Resin withBoc-D-Ala-benzhydrylaminopolystyrene-1%-divinylbenzene resin preparedfrom benzhydrylaminopolystyrene-1% divinylbenzene resin (Beckman Inst),Boc-D-Ala-OH, DIC and HBT, was obtained the corresponding D-Ala₁₀analogs of LHRH:

N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Nal(2)-Ser-Tyr-D-Iph-Leu-Arg-D-AlaNH₂, and

N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Dih-Leu-Arg-Pro-D-AlaNH₂,

N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Dmh-Leu-Arg-Pro-D-AlaNH₂,

N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Deh-Leu-Arg-Pro-D-AlaNH₂,

N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Dhh-Leu-Arg-Pro-D-AlaNH₂,

N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Mpa-Leu-Arg-Pro-D-AlaNH₂,

N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Pia-Leu-Arg-Pro-D-AlaNH₂,

N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Ppa Leu-Arg-Pro-D-AlaNH₂, and

N-Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Bpa-Leu-Arg-Pro-D-AlaNH₂.

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-Deh-Leu-Arg-Pro-D-AlaNH₂, [α]_(D)²⁵ -21.6° (CO.4, (HoAc),

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Deh-Leu-Arg-Pro-D-AlaNH₂,

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Dmh-Leu-Arg-Pro-D-AlaNH₂,

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Dhi*-Leu-Arg-Pro-D-AlaNH₂,

N-AC-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Dph-Leu-Arg-Pro-D-AlaNH₂,

N-Ac-D-Nal(2)-D-pf-Phe-D-Trp-Dev-Tyr-D-Deh-Leu-Arg-Pro-D-AlaNH₂, and

N-Ac-D-Nal(2)-D-pF-Phe-D-Trp-Ser-Tyr-D-Dmh-Leu-Arg-Pro-D-AlaNH₂.

EXAMPLE 2

For the synthesis of analogues with a C-terminal Pro-NHCH₂ CH₃, asynthesis program identical to that described in Example 1 was used. TheBeckman 990 Synthesizer reaction vessel was loaded with 2.13 g. ofBoc-Pro-O-Resin, prepared by the reaction of equimolar ratios of the drycesium salt of Boc-Pro-OH with chloromethyl-polystyrene/1%divinylbenzene (Lab Systems, Inc.). The quantity of Boc-Pro-O-Resintaken contained 1.4 mmol. of proline.

The resin was coupled sequentially with a 2.0 to 2.5 molar excess ofeach protected amino acid and DCC. Thus, the resin was reacted duringsuccessive coupling cycles with

1.61 g. Boc-Arg(Tosyl)-OH,

0.93 g. Boc-Leu-OH H₂ O,

0.94 g. Boc-N,N'-guanido-diisopropylhomoarginine,

0.49 g. 1-hydroxybenzotriazole,

1.75 g. N-Boc-O-2-bromobenzyloxycarbonyl-L-tyrosine, and

1.11 g. Boc-Ser(Benzyl)-OH.

At this point in the synthesis the quantity of protected polypeptideresin was split in half and one half was carried through to completionby sequential reaction with

0.57 g. Boc-D-Nal(2)-OH,

0.480 g. Boc-D-p-F-Phe-OH

0.21 g. N-Ac-L-proline.

The resin was removed from the reaction vessel, washed with CH₂ Cl₂, anddried in vacuo to yield 2.26 g. of protected polypeptide resin.

The protected polypeptide was cleaved from the resin by aminolysis with25 mL. of ethylamine for 18 hours at 2° C. The ethylamine was allowed toevaporate and the resin was extracted with methanol. The methanol wasevaporated to yield 1.39 g. ofN-Ac-L-Pro-D-p-Cl-Phe-Trp-Ser(Benzyl)-Tyr(2,6-dichlorobenzyl)-D-Dih-Leu-Arg(Tosyl)-Pro-NHCH₂CH₃. This protected peptide was mixed with 3 ml of anisole and 30 mL.redistilled (from CoF₃) anhydrous liquid HF at 0° C. for 30 minutes in aKel-F reaction vessel. The HF was evaporated under vacuum and theresidue was washed with ether. The residue was dissolved in 2 M aceticacid and lyophilized to yield 0.82 g. of crudeN-Ac-L-Pro-D-p-F-Phe-Trp-Ser-Tyr-D-Dih-Leu-Arg-Pro-NHCH₂ CH₃ as itsacetic acid addition salt. Final purification was achieved bypreparative high performance liquid chromatography of a 20 mg. sample ona 2.5×100 mm. column of 40-50 microns. octadecylsilylated silica (Merck,Lichroprep C₁₈).

Proceeding in a similiar manner, but substituting the appropriateprotected amino acid residues where appropriate, there are prepared thefollowing compounds:

N-Ac-D-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dih-Leu-Arg-Pro-NHEt,

N-Ac-D-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dph-Leu-Arg-Pro-NHEt,

N-Ac-D-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dmh-Leu-Arg-Pro-NHEt,

N-Ac-D-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Deh-Leu-Arg-Pro-NHEt,

N-Ac-D-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dhh-Leu-Arg-Pro-NHEt,

N-Ac-D-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Prh-Leu-Arg-Pro-NHEt,

N-Ac-D-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Hha-Leu-Arg-Pro-NHEt,

N-Ac-D-Nal(2)-D-p-F-Phe-D-Trp-Ser-Tyr-D-Dpa-Leu-Arg-Pro-NHEt,

Repeating the above cleavage, substituting a stoichiometric amount ofmethylamine and propylamine for ethylamine there are obtained thecorresponding methylamide or propylamine of the aforementionednonapeptide.

EXAMPLE 3

A. A solution of 0.1 g of the hydrogen fluoride salt ofN-Ac-L-Pro-D-p-F-Phe-Trp-Ser-Tyr-D-Dia Leu-Arg-Pro-GlyNH₂ (SeeExample 1) is dissolved in 50 mL of water and passed through a column of50 g Dowex 3 anion exchange resin which had previously been equilibratedwith acetic acid and washed with deionized water. The column is elutedwith deionized water and the effluent is lyophilized to yield thecorresponding acetic acid salt ofN-Ac-L-Pro-D-p-F-Phe-Trp-Ser-Tyr-D-Dia-Leu-Arg-Pro-Gly-NH₂, [α]_(D) ²⁵-15.4° (C 1, HOAc

Repeating the above, substituting other acids for acetic acid during theequilibration of the resin, there may be obtained, for example, thecorresponding salts with hydrochloric acid, hydrobromic acid, sulfuricacid, phosphoric acid, nitric acid, benzoic acid, and the like.

Similarly there may be prepared the acid addition salts of the otherpeptides analogous to LHRH, described herein.

B. In the case of salts of low water solubility, these may be preparedby precipitation from water utilizing the desired acid. For example:

Zinc tannate salt--a solution of 10 mg ofN-Ac-L-Pro-D-p-Cl-Phe-D-Nal(2)-Ser-Tyr-D-Dih-Leu-Arg-Pro-GlyNH₂ aceticacid salt in 0.1 mL of water was treated with a solution of 8 mg oftannic acid in 0.08 mL of 0.25 M NaOH. A solution of 5 mg of ZnSO₄heptahydrate in 0.1 mL of water was immediately added to the solution ofthe LHRH analogue.

The resultant suspension was diluted with 1 mL water and the precipitatewas centrifuged. The supernatant was decanted and the residue was washedtwice with 1 mL portions of water by centrifugation of the precipitateand decantation of the supernatant. The precipitate was dried in vacuoto yield 15 mg of the mixed zinc tannate salt of the above named LHRHanalogue.

Pamoate salt--to a solution of 50 mgN-Ac-L-Pro-D-p-F-Phe-D-Nal(2)-Ser-Tyr-D-Dih-Leu-Arg-Pro-Gly-NH₂ aceticacid salt in a mixture of 1.6 mL of ethanol and 0.1 mL of 0.25 M NaOHwas added solution of 11 mg of pamoic acid in 0.3 mL of 0.25 M NaOH. Thesolvents were removed at reduced pressure and the residue was suspendedin 2 mL of water, centrifuged, and the supernatant was decanted. Theprecipitate was washed with 1.5 mL H₂ O, centrifuged, and thesupernatant was decanted. The precipitate was dried in vacuo to yield 54mg of the pamoate salt of the above named LHRH analogue.

In a similar manner other salts of low water solubility may be prepared.

C. Preparation of acid addition salt from free peptide.

To a solution of 50 mg ofN-Ac-L-Pro-D-p-F-Phe-D-Nal(2)-Ser-Tyr-D-Dia-Leu-Arg-Pro-GlyNH₂ as thefree base is added 30 mL of 1N acetic acid. The resulting solution islyophilized to yield 50 mg. of the acetic acid salt of the above [α]_(D)²⁵ -515.4° (C 0.5, HOAc)

Similarly, replacing acetic acid with other acids (in stoichiometricallyequivalent amounts relative to peptide) there was obtained other acidadditon salts of the peptides herein, for example, the salts withhydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid,nitric acid.

D. Preparation of salt with metal cation, e.g., zinc salt.

To a solution of 50 mgN-Ac-L-Pro-D-p-F-Phe-D-Nal(2)-Ser-Tyr-D-Dih-Leu-Arg-Pro-Gly-NH₂ aceticacid salt in a mixture of 0.4 mL of 0.25 M NaOH, 0.3 mL water, and 1 mLethanol was added a solution of 15 mg of ZnSO₄ heptahydrate in 0.2 ml ofwater. The precipitate was centrifuged and the supernatant was decanted.The precipitate was washed with 1 mL of water by centrifugation anddecantation of the supernatant. The precipitate was dried in vacuo toyield the zinc salt of the above named LH-RH analogue.

In a similar manner salts with other multivalent cations e.g. calcium,bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmiumand the like, may be prepared.

EXAMPLE 4

A solution of 50 mg ofN-Ac-L-Pro-D-p-F-Phe-D-Nal(2)-Ser-Tyr-D-Dih-Leu-Arg-Pro-Gly-NH₂ aceticacid salt in 25 ml. of water is passed through a 50 g column of Dowex 1(strongly basic, quaternary ammonium anion exchange resin) which hadbeen equilibrated with NaOH solution to make the counter ion hydroxide.The column is eluted with 150 ml of water and the eluant is lyophilizedto yield 45 mg of the corresponding polypeptide as the free base.

Similarly other acid addition salts of compounds of the peptides herein,e.g., those mentioned in Example 6, may be converted to thecorresponding free bases.

EXAMPLE 5

The following are typical pharmaceutical compositions containing, asactive ingredient, an LHRH antagonist of the present invention, forexample N-Ac-L-Pro-D-p-F-Phe-D-Nal(2)-Ser-Tyr-D-Dih-Leu-Arg-Pro-GlyNH₂,by itself or as a pharmaceutically acceptable salt, e.g., the aceticacid addition salt, the zinc salt, the zinc tannate salt, etc.

A. Tablet formulations for buccal (e.g. sublingual) administration:Polymer Microcapsules

    ______________________________________                                        1.     LHRH Antagonist       10.0   mg                                               Compressible Sugar, USP                                                                             86.0   mg                                               Calcium Stearate      4.0    mg                                        2.     LHRH Antagonist       10.0   mg                                               Compressible Sugar, USP                                                                             88.5   mg                                               Magnesium Stearate    1.5    mg                                        3      LHRH Antagonist       5.0    mg                                               Mannitol, USP         83.5   mg                                               Magnesium Stearate, USP                                                                             1.5    mg                                               Pregelatinized Starch, USP                                                                          10.0   mg                                        4.     LHRH Antagonist       10.0   mg                                               Lactose, USP          74.5   mg                                               Pregelatinized Starch, USP                                                                          15.0   mg                                               Magnesium Stearate, USP                                                                             1.5    mg                                        ______________________________________                                    

Method of Manufacture

a. LH-RH Antagonist is dissolved in water, a sufficient quantity to forma wet granulation when mixed with the sugar portion of the excipients.After complete mixing, the granulation is dried in a tray or fluid-beddryer. The dry granulation is then screened to break up any largeaggregates and then mixed with the remaining components. The granulationis then compressed on a standard tabletting machine to the specifictablet weight.

b. In this manufacturing method, all formulations would include 0.01%gelatin, USP. The gelatin would be first dissolved in the aqueousgranulation solvent followed by the LH-RH analog. The remaining stepsare as in (a) above.

Formulation 4 could also be used as a tablet for oral administration.

B. Long Acting intramuscular injectable formulation.

1. Long Acting I.M. Injectable--Sesame Oil Gel

    ______________________________________                                        LHRH Antagonist         10.0   mg                                             Aluminum monostearate, USP                                                                            20.0   mg                                             Sesame oil q.s. ad      1.0    ml                                             ______________________________________                                    

The aluminum monostearate is combined with the sesame oil and heated to125° C. with stirring until a clear yellow solution forms. This mixtureis then autoclaved for sterility and allowed to cool. The LH-RH analogueis then added aseptically with trituration. Particularly preferred LH-RHanalogues are salts of low solubility, e.g. zinc salts, zinc tannatesalts, pamoate salts, and the like. These exhibit exceptionally longduration of activity.

2. Long Acting I.M. Injectable--Biodegradable Polymer Microcapsules

    ______________________________________                                        LHRH Antagonist     1%                                                        25/75 glycolide/lactide                                                                          99%                                                        copolymer (0.5 intrinsic                                                      viscosity)                                                                    ______________________________________                                    

Microcapsules (0-150*) of above formulation suspended in:

    ______________________________________                                        Dextrose         5.0%                                                         CMC, sodium      0.5%                                                         Benzyl alcohol   0.9%                                                         Tween 80         0.1%                                                         Water, purified q.s.                                                                           100.0%                                                       ______________________________________                                    

25 mg of microcapsules would be suspended in 1.0 ml of vehicle.

C. Aqueous Solution for Intramuscular Injection

    ______________________________________                                        LHRH Antagonist         500    mg                                             Gelatin, nonantigenic   5      mg                                             Water for injection q.s. ad                                                                           100    ml                                             ______________________________________                                    

Dissolve gelatin and LHRH antagonist in water for injection, thensterile filter solution.

D. Formulation for Rectal Administration Suppository Vehicle for RectalAdministration

    ______________________________________                                        LHRH Antagonist       5.0    mg                                               Witepsol H15          20.0   gm                                               ______________________________________                                    

The LHRH antagonist is combined with the molten Witepsol H15, mixed welland poured into 2 gm molds.

What is claimed is:
 1. A compound which isN-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Deh-Leu-Arg-Pro-D-AlaNH₂.